Cloned (Comment) | Organism |
---|---|
gene adhB, expression of mutant I86A in Escherichia coli HB101 (DE3) as protein C-terminally tagged with two methionines followed by Leu-Glu-His6 | Thermoanaerobacter ethanolicus |
Crystallization (Comment) | Organism |
---|---|
purified recombinant His-tagged mutant I86A, hanging drop vapour diffusion, mixing of 0.002 ml of 8 mg/ml protein in 50 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.6, 20 mM NaCl, 0.1 mM dithiothreitol, 0.1 mM ethylenediaminetetraacetic acid, and 2 mM (R)-1-phenylethanol, with 0.2% D-fructose 1,6-disphosphate trisodium salt octahydrate, 0.2% glycerol phosphate disodium salt hydrate, 0.2% O-phospho-L-serine, 0.2% O-phosphol-L-styrosine, 0.2% phytic acid sodium salt hydrate, 0.02 M HEPES sodium pH 6.8, and 0.002 ml reservoir solution containing 20% v/v PEG 400, 0.1 M Na HEPES, pH 7.0, room temperature, 6-12 h, X-ray diffraction structure determination and analysis, molecular replacement, modeling | Thermoanaerobacter ethanolicus |
Protein Variants | Comment | Organism |
---|---|---|
I86A | site-directed mutagensis, the secondary alcohol dehydrogenase I86A mutant is stereospecific for (R)-alcohols instead of (S)-alcohols, in contrast to the wild-type enzyme, the mutation I86A allows large substituents to fit into the large pocket of I86ATeSADH, which corresponds to the small pocket in wild-type TeSADH, modeling of the stereopreference of TeSADH I86A | Thermoanaerobacter ethanolicus |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
37590 | - |
4 * 37590, sequence calculation, a tetramer formed by a dimer of dimers | Thermoanaerobacter ethanolicus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-1-phenylethanol + NAD+ | Thermoanaerobacter ethanolicus | activity of mutant I886A, the wild-type enzyme is (S)-specific forming (S)-1-phenylethanol | acetophenone + NADH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermoanaerobacter ethanolicus | P77990 | gene adhB, mutant I86A | - |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally His-tagged mutant I86A from Escherichia coli HB101 (DE3) by nickel affinity chromatography | Thermoanaerobacter ethanolicus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-1-phenylethanol + NAD+ | activity of mutant I886A, the wild-type enzyme is (S)-specific forming (S)-1-phenylethanol | Thermoanaerobacter ethanolicus | acetophenone + NADH + H+ | - |
r |
Subunits | Comment | Organism |
---|---|---|
tetramer | 4 * 37590, sequence calculation, a tetramer formed by a dimer of dimers | Thermoanaerobacter ethanolicus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Thermoanaerobacter ethanolicus | |
NADH | - |
Thermoanaerobacter ethanolicus |
General Information | Comment | Organism |
---|---|---|
additional information | the mutation I86A allows large substituents to fit into the large pocket of I86ATeSADH, which corresponds to the small pocket in wild-type TeSADH, modeling of the stereopreference of TeSADH I86A | Thermoanaerobacter ethanolicus |